The calcium sensor CBL7 is required for Serendipita indica‐induced growth stimulation in Arabidopsis thaliana, controlling defense against the endophyte and K+ homoeostasis in the symbiosis

Abstract Calcium is an important second messenger in plants. The activation of Ca2+ signalling cascades is critical in the activation of adaptive processes in response to environmental stimuli. Root colonization by the growth promoting endophyte Serendipita indica involves the increase of cytosolic Ca2+ levels in Arabidopsis thaliana. Here, we investigated transcriptional changes in Arabidopsis roots during symbiosis with S. indica. RNA‐seq profiling disclosed the induction of Calcineurin B‐like 7 (CBL7) during early and later phases of the interaction. Consistently, reverse genetic evidence highlighted the functional relevance of CBL7 and tested the involvement of a CBL7‐CBL‐interacting protein kinase 13 signalling pathway. The loss‐of‐function of CBL7 abolished the growth promoting effect and affected root colonization. The transcriptomics analysis of cbl7 revealed the involvement of this Ca2+ sensor in activating plant defense responses. Furthermore, we report on the contribution of CBL7 to potassium transport in Arabidopsis. We analysed K+ contents in wild‐type and cbl7 plants and observed a significant increase of K+ in roots of cbl7 plants, while shoot tissues demonstrated K+ depletion. Taken together, our work associates CBL7 with an important role in the mutual interaction between Arabidopsis and S. indica and links CBL7 to K+ transport.

the infection, the controlled reduction of plant defence responses becomes paramount to facilitate the establishment of the mutual interaction between the endophyte and its host plant. In this respect, balancing of plant hormone contents and the tight control of indole glucosinolates are reported to play essential roles (Lahrmann et al., 2015;Nongbri et al., 2012;Xu et al., 2018).
The elevation of cytosolic Ca 2+ concentrations in Arabidopsis root cells through the influx of Ca 2+ via the Cyclic nucleotide gated channel 19 represents a further critical asset in consolidating the plant−fungus interaction (Jogawat et al., 2020;Vadassery & Ranf, Drzewiecki, et al., 2009). Calcium is an essential plant macronutrient that plays an important role in plant growth and development. At the same time, Ca 2+ serves as an important second messenger in plants that is involved in orchestrating adequate responses to external signals, including biotic stresses (Thor, 2019). Cytosolic Ca 2+ concentrations show highly dynamic and specific spatiotemporal patterns, which are governed by the type and intensity of the perceived stimulus (Pivato & Ballottari, 2021). Depending on the particular stimulus, plant cells respond by producing specific Ca 2+ signatures that differ in their frequency, amplitude and duration (Batistič & Kudla, 2012). To decipher the different Ca 2+ signatures, plants possess a broad set of different sensor molecules that either directly modify target proteins through phosphorylation or act through their physical interaction with specific partner proteins, including protein kinases (Kudla et al., 2018). This diverse set of Ca 2+ sensor molecules encompasses Calmodulins (CaMs), Calmodulin-like proteins (CMLs), Ca 2+ -dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs), as well as their interacting kinases (CIPKs).
The latter forming a two-component system in which the CBLs act as Ca 2+ sensors that relay their stimulation to specific CIPKs, which subsequently interact with downstream target proteins, such as nutrient transporters or ion channels (Liu & Tsay, 2003;Maierhofer et al., 2014;Ragel et al., 2015). In particular, the regulation of the high affinity potassium transporter HAK5 and the highly selective potassium channel AKT1 by CIPK23 is well documented (Lan et al., 2011;Lee et al., 2007;Ragel et al., 2015). However, there is also evidence for an interaction of CIPKs with diverse components of the abscisic acid response pathway, including the corepressors ABI1 and ABI2 (Ohta et al., 2003). The Arabidopsis genome contains 10 CBL and 26 CIPK genes. Among each other, the CBLs and CIPKs form functional complexes and it is noteworthy that CBLs are not promiscuously interacting with all CIPKs, but show preferences and interact only with a specific subset of CIPKs to facilitate efficient signal transduction and integration (Kudla et al., 2010). Overall, the specific combination of CBL-CIPK modules is the key to provide versatility and flexibility in the regulation of a multitude of external stimuli that marshal ion transport in plants (Tong et al., 2021). In addition to the regulation of K + transporters, CBL-CIPK interactions have recently been described to control the polarization of plasma membranes (Yang et al., 2019). In this manner, CBL-CIPKs can also indirectly regulate voltage-gated K + channels, such as the outwardrectifying K + channels SKOR and GORK (Dreyer et al., 2004).
Up to date, the functional role of Calcineurin B-like 7 (CBL7) is only partially elucidated. A recent study associates CBL7 with the regulation of plant responses towards low nitrate in Arabidopsis (Ma et al., 2015). The work highlights a substantial expression of CBL7 in root tissues and its induction under nitrogen and nitrate limiting conditions. Moreover, the authors report on the involvement of CBL7 in the transcriptional regulation of two high-affinity nitrate transporter genes, NRT2.4 and NRT2.5, without providing evidence for a molecular mechanism that could explain how the downstream genes are targeted. It is speculated that the localization of CBL7 to the nucleus facilitates its interaction with nitrate-starvation response-related transcription factors (TFs), such as NLP7 (Kiba & Krapp, 2016;Konishi & Yanagisawa, 2013;Krouk & Kiba, 2020;Marchive et al., 2013).
In this study, we identified the Ca 2+ sensor CBL7 as an essential molecular component in the interaction between the root colonizing endophyte S. indica and A. thaliana. CBL7 is consistently induced upon root infection with the fungus, not only at early stages of the infection, but also at later phases. Furthermore, we were able to demonstrate that the availability of CBL7 is crucial for the development of the fungus-mediated plant growth promotion and for the proper distribution of potassium in the plant. The comprehensive transcriptomics analysis of the cbl7 mutant grown with and without the fungus in comparison to corresponding wildtype plants additionally pinpointed a role of CBL7 in harmonizing plant defense responses for the long-term interaction between the endophyte and Arabidopsis. Taken together, our results establish CBL7 as a novel key component required for the successful establishment of the symbiosis between S. indica and A. thaliana.

| Root growth promotion assay
Surface-sterilized Arabidopsis seeds were plated on vertical ½ MS plates. After stratification (2 days at 4°C), the plates were transferred to the growth chamber and the seedlings were grown vertically for 1 week. Thereafter, four to five seedlings were transferred to Petri dishes containing solidified Plant Nutrition Medium (PNM) supplemented with 50 mM NaCl (Johnson et al., 2013). Each seedling was then associated with a 5 mm Ø medium plug extracted from either sterile AP plates (control) or from AP plates harbouring a 1-week-old S. indica mycelium (cocultivation).
The PMN plates with the control seedlings and the seedlings cocultivated with the fungus were further kept in a growth chamber maintained at 23.5°C, 16/8 h photoperiod, 100 µmol photons m −2 s −1 light intensity for another 10 days. After that time, the plants were photographed for the further analysis of the root system and the plant material was either used for RNA extraction or the determination of the fresh weight.

| Quantitative analysis of root system architecture traits
The stimulation of root growth is a well-described trait in the interaction of S. indica with its host plant. With the aim to quantify the effect of S. indica in the different genotypes and treatments, respectively, photographs of the plates were captured with a digital camera at a fixed distance of 29 cm. Using Adobe Photoshop CC, the images were cropped to a height of 14 cm maintaining only the part containing the root system and converted to black and white images.

| qPCR analysis
Real-time quantitative RT-PCR was conducted as previously described (Pérez-Alonso et al., 2021). In brief, total RNA from three different biological samples was converted into cDNA using M-MLV reverse transcriptase and oligo(dT) 15 primer. Two nanograms of cDNA was then used as template for the qPCR reactions, which were conducted in triplicate (technical replicates). The oligonucleotide pairs used in the experiments are given in Supporting Information: Data Sheet 1. The reactions were monitored on a Lightcycler 480 Real-time PCR system (Roche Diagnostics). Differential gene expression in Arabidopsis was analysed by using the comparative 2 C -ΔΔ t method (Livak & Schmittgen, 2001) with Adenine phosphoribosyl transferase 1 (APT1, At1g27450) as reference gene (Jost et al., 2007). Root colonization with S. indica was monitored with a primer pair for fungal translation elongation factor EF-1α (TEF1) (Bütehorn et al., 2000). The fungal TEF1 cDNA levels were expressed relative to the plant Glycerinaldehyde-3-phosphate dehydrogenase C2 (GAPC2, At1g13440) cDNA levels. To exclude that the amplified DNA fragments stem from DNA of dead fungal tissues within the roots, all data presented here derived from cDNA libraries generated from RNA of colonized roots.

| Trypan blue staining of fungal hyphae and spores
To visually inspect root colonization, 10−12 small root samples from control and cocolonized plants were employed. First, the root samples were thoroughly washed with deionized water. Next, the root samples were cut in 1 cm long pieces and incubated overnight in 10 N KOH. The root samples were then rinsed five times with sterile H 2 O, before they were incubated for 5 min in 0.1 N HCl. Finally, the samples were incubated in a 0.05% trypan blue solution (w/v), before they were partially decolorized with lactophenol over 10 min. Before the specimen were mounted on glass slides and examined by microscopy, they were washed once with 100% ethanol and three times with sterile H 2 O and stored in 60% glycerol (v/v).

| K + quantification
The analysis of endogenous cation contents of infected and control plants was performed according to Conchillo et al. (2021) in fractions of root and shoot samples. In brief, the fungi were first K + depleted buy inoculating 100 ml AP medium containing 1 mM K + with 1.5 × 10 3 of the corresponding spores and incubating it for 1 week at 28°C. Next, the mycelia were harvested by centrifugation and transferred to fresh AP medium without K + .
After another week of incubation, the K + starved spores were harvested. At the same time, Arabidopsis seedlings were germinated on ½ MS plates for 1 week. Thereafter, the seedlings were

| CBL7 is induced in the symbioses between Arabidopsis and S. indica
The growth promoting effect of S. indica on multiple host plant species has already been well-characterized (Mensah et al., 2020).
However, the precise molecular mechanism by which the fungal root GO term classifications. In addition, it provided evidence for the enrichment of genes related to a group of GO terms associated with cellular Ca 2+ homoeostasis and Ca 2+ signalling processes, which attracted our attention and on which we followed up in this study.
To gain a better understanding of the role of Ca 2+ -related processes in the mutual interaction between S. indica and Arabidopsis, we extracted the relative expression profiles of 88 Ca 2+ signalling-related genes from the nonfiltered RNA-seq data sets and performed a hierarchical cluster analysis (Supporting Information: Image 1B). We have been particularly intrigued by the expression profile of the genes in Cluster 1, which contained the four genes High affinity K + transporter 5 (HAK5), Calmodulin-like 4 (CML4), CIPK13 and CBL7. The genes were consistently induced both at early and later stages of the plant−fungus interaction, which could be confirmed by qRT-PCR analysis (Figure 1c). Activation of these genes might therefore be required both for the establishment and preservation of the mutual plant−fungus interaction.

| CBL7 is required for fungus-mediated growth promotion
CBL proteins are plant-specific Ca 2+ sensors that are predicted to decode calcium transients through interaction with CIPKs (Tang et al., 2020). It has previously been shown that several CBL proteins, that is, CBL1, CBL8, CBL9 and CBL10, interact with CIPK23 to regulate HAK5 transport activity (Ragel et al., 2015). Hence, we speculated that CBL7 and CIPK13 may physically interact with each other to control HAK5 activity as their downstream target, because of their shared expression profiles in response to the infection of Arabidopsis seedlings with S. indica. To test this hypothesis, we took a reverse genetics approach to assess the role of CBL7, CIPK13 and HAK5 in the fungus-mediated promotion of plant growth. To do so, we used two independent T-DNA insertion mutants for both CBL7 and CIPK13, as well as the previously described hak5 mutant. The mutants were grown for 7 days on ½ MS medium alongside with corresponding wild-type control plants, before the seedlings were transferred to PNM medium, where they were either cocultivated with S. indica or a mock control. After 10 days, the vertically grown plants were photographed (Supporting Information: Figure S2), and reported (Ma et al., 2015). Additionally, we again found that the highaffinity potassium transporter gene HAK5 was significantly induced (log 2 FC = 1.85, p adj. = 0.022). Although our previous results questioned the role of HAK5 in the plant−fungus interaction, the reiterated appearance of HAK5 led us to analyse the potassium content in roots and shoots of cbl7-2 and Col-3 to determine whether CBL7 could be involved in the regulation of potassium homoeostasis in Arabidopsis. For this, the K + contents of fungus and mock-treated mutant and wt seedlings were quantified by atomic emission spectroscopy. As shown in Figure 4, the potassium content of the cbl7-2 roots grown at 0 mM KCl was similar to that of the Col-3 control plants. However, the previously described decrease in K + contents in response to the inoculation with S. indica (Conchillo et al., 2021) was only observed in control plants, but not in cbl7-2.
Interestingly, the K + level in the shoots of cbl7-2 plants was significantly lower than in the mock-treated wild type, equal to the level observed for Col-3 treated with the fungus. Although K + levels showed a tendency to decrease in cbl7-2 shoots, there was no significant difference between mock and fungus-treated cbl7-2 plants. Plants grown on plates containing 1 mM KCl showed a similar picture (Figure 4b), only wild-type Col-3 plants showed the expected decrease in K + in response to the S. indica infection. Furthermore, we found a significant increase of K + in cbl7-2 roots, while the K + content in cbl7-2 shoots appeared to be significantly reduced. We have therefore concluded that CBL7 contributes to the regulation of the distribution of K + in the plant. The functional loss of CBL7 is unequivocally linked to an altered K + distribution profile, which is characterized by an increase of K + in the roots, most likely due to a lack of transport of K + to areal plant tissues. At the same time, our data suggest that CBL7 could also play a key role in the observed decrease in K + levels in plants challenged with S. indica, as cbl7 mutants present a reduced reduction of K + when cocultivated with S. indica.

| The cbl7 mutant shows increased plant defense responses
The above results highlighted the transcriptional regulation of   (PAD3), which are known to participate in glucosinolate and camalexin biosynthesis (Frerigmann et al., 2016;Glawischnig, 2007). Furthermore, we found an induction of the myrosinase genes BGLU34 and BGLU35 that are involved in the turnover of glucosinolates (Wittstock & Burow, 2010). The observation of an induction of plant defense-related compounds is further supported by the induction of TFs NAC042, WRK33 and WRK51, which have been linked with the regulation of camalexin and indole glucosinolate biosynthesis (Birkenbihl et al., 2012;Frerigmann & Gigolashvili, 2014;Saga et al., 2012;Zhou et al., 2020). Furthermore, we also observed an induction of WRKY70, a TF involved in modulating cell wall-related defense responses (Li et al., 2017). Moreover, the functional analysis of the transcriptomics data revealed an enrichment of glutathione S-transferases among the induced genes in cbl7-2, which included the genes GSTU10, GSTU12, GSTF3, GSTF6 and GSTF7. Glutathione S-transferases are readily induced by a wide range of stress conditions, including bacterial and fungal infections (Dixon et al., 2002;Gullner et al., 2018). Considering that all these processes are observed in the cbl7 loss-of-function mutant, it must be concluded that CBL7 is involved in the suppression of multilayered defense responses to facilitate the establishment of S.
indica in the root apoplast. However, other processes that appear enriched in S. indica challenged wt plants but not in cbl7, such as the induction of sucrose transporter genes and WRKY46 orchestrated abiotic stress responses (Chen et al., 2010(Chen et al., , 2012Ding et al., 2015), or the repression of bHLH100 and MYB72 mediated metal ion homoeostasis (Sivitz et al., 2012;Zamioudis et al., 2015), are likely to contribute to the significant difference in growth promotion between cbl7 and wt triggered by the fungus.
Based on our hypothesis of an increased defense response in the cbl7 knockout mutant, it must be expected that the roots of mutant

| DISCUSSION
Calcium signalling plays an important role in the regulation of a wide array of biological processes in eukaryotic physiology, including pathogenic and beneficial plant−microbe interactions (Vadassery & Oelmüller, 2009). In this study we showed that CBL7 is among the very few genes that display a consistent positive transcriptional response in Arabidopsis wild-type plants infected with the beneficial root endophyte S. indica, both at early and later stages of the interaction (Figure 1). The coexpression of CBL7, CIPK13 and HAK5 prompted us to speculate that there might be a relevant connection between these components, which could regulate the uptake of potassium into host plants and, thus, their nutrition with this essential macronutrient. The activation of HAK5 through phosphorylation by CIPK23 has previously been described for Arabidopsis and tomato plants (Amo et al., 2021;Ragel et al., 2015). Hence, an interaction cascade between the three components appeared possible. However, when we analysed the relevance of the different components with respect to their impact on the growth promoting effect on Arabidopsis roots, we had to realize that only CBL7, but neither CIPK13 nor HAK5 interfered with growth promotion (Figure 2).
While both cbl7 mutant alleles showed a severe loss of growth promotion, none of the other investigated mutants demonstrated significant differences to wt. Although CIPKs and potassium transporters form large gene families, which opens the possibility that their loss might be compensated by other family members, it must be concluded that CIPK13 and HAK5 are not critical for the fungus-triggered plant growth promotion. The possible plastid localization of CIPK13 further argues against an interaction with CBL7 in vivo (Schliebner et al., 2008).
Previous work on the role of CBL7 highlighted its interaction with the A. thaliana plasma membrane proton ATPase 2 (AHA2) (Yang et al., 2019). In their model, the CBL7/AHA2 complex is further stabilized by the interaction with protein kinase SOS2-like 5 (PKS5), also referred to as CIPK11. The authors suggest that the Ca 2+mediated dissociation of the CBL7/CIPK11/AHA2 complex under saline-alkali stress conditions translates into the activation of AHA2.
Activation of AHA2, in turn, leads to hyperpolarization of the plasma membrane, which is likely to affect the transport activity of Shakerlike K + channels in the root. SKOR, an outward-rectifying K + channel, is reported to be crucial for the loading of K + into the xylem and, thus, the long-distant transport of K + within the plant (Gaymard et al., 1998). Moreover, SKOR is known to form heteromeric outwardrectifying K + channel units with a second Shaker-like channel, GORK (Dreyer et al., 2004). SKOR and GORK facilitate K + transport only when the plasma membrane is depolarized (Dreyer & Blatt, 2009).
A recent publication further pinpointed the importance of the complex interplay between different nutrient transporter systems and proton pumps in nutrient cycling in plants (Dreyer, 2021). Based on these observations, it must be assumed that a loss of CBL7 will likely result in a disrupted regulation of AHA2 activity and, consequently, a hyperpolarization of the plasma membrane, which entails a reduced transport activity of SKOR and GORK. As a result, the assimilated K + would accumulate in the roots, and only reduced amounts of K + would reach the shoot. In fact, our analysis of K + levels in wild-type and cbl7 mutants corroborates this hypothesis (Figure 3).
The cbl7 mutant plants showed a pronounced increase of K + contents in the roots, which we attribute to possible impairment of K + xylem transport. Potassium depletion in the shoot could trigger the induction of HAK5 in cbl7, as multiple independent studies demonstrated that HAK5 is induced under K + starvation (Ahn et al., 2004;Armengaud et al., 2004;Gierth et al., 2005;. A recent study demonstrated the induction of HAK5 in the host plant as a general consequence of the symbiotic interaction (Conchillo et al., 2021), which would also explain why we found HAK5 under the consistently induced genes in wild-type plants ( Figure 1). Furthermore, the latter study demonstrated that the In this context, it needs to be remarked that the expression of neither SKOR and NPF7.3/NRT1.5 nor SGN3, which have previously been associated with K + transport in Arabidopsis (Drechsler et al., 2015;Pfister et al., 2014), appeared to be significantly altered in the cbl7-2 mutant. It will be a thrilling future task to decipher whether impaired repression of plant defense responses or out-of-control potassium translocation in cbl7 are the cause of the observed reduced root colonization and missing fungus-triggered plant growth promotion.

ACKNOWLEDGMENTS
The authors are grateful to Mar González Ceballos for her excellent technical support. Moreover, we acknowledge the critical discussion of our work with experts in the field, including Ingo Dreyer